Introduction

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Atopic dermatitis is a chronically relapsing inflammatory skin disease characterized by episodes of intense pruritus, multiple lesions with erythema, excoriation, erosions, lichenification, papules, dry skin, and susceptibility to cutaneous infection. Histopathological studies shown that the skin lesions of atopic dermatitis are characterized by acanthosis and spongiosis in the epidermis, predominant infiltration by CD1⁺ cells and activated CD4⁺ T cells in the dermis, and extensive deposition of eosinophil granule proteins, such as eosinophil major basic protein and eosinophil cationic protein (Leiferman et al., 1985; Bruijnzeel et al., 1993). In addition, serum levels of eosinophil cationic protein have been reported to correlate with the severity of disease (Tsuda et al., 1992).

The study of atopic dermatitis has been hampered by the lack of appropriate experimental models; however, spontaneously occurring dermatitis in mice has recently been reported as a model (Matsuda et al., 1997). In humans, epicutaneous application of aeroallergens, commonly referred to as the atopy patch test, can provoke eczematous skin reactions in atopic dermatitis patients (Clark and Adinoff, 1989). The atopy patch test has been proposed as an in vivo model for the study of allergic inflammation in atopic dermatitis (Langeveld-Wildschut et al., 1996) because the reactions to the atopy patch test resemble atopic dermatitis lesions in terms of both macroscopic appearance and histological characteristics (Bruijnzeel-Koomen et al., 1988). On the other hand, intracutaneous administration of allergens induces a so-called late-phase reaction (Kaliner, 1984), which is limited to the dermis and do not induce eczema-like changes in the epidermis (Dolovich et al., 1973; Charlesworth et al., 1989a; Bos et al., 1994).

The differences between the atopy patch test reaction and late-phase skin reaction led to the hypothesis that epicutaneous exposure to protein allergens plays a role in the development of eczema in atopic dermatitis. According to the hypothesis, we developed a new animal model in which eczema-like lesions develop when an antigen is applied topically by patch to epicutaneously sensitized guinea pigs.

We tested a number of compounds for an inhibitory effect on allergic inflammation in the guinea pig model of eczema and discovered that some imidazopyridazine derivatives had an inhibitory effect. Among them, we selected TAK-427 (Fig. 1) as a candidate compound.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of TAK-427.

In this article, we describe histopathologic and pharmacological characteristics of the damaged skin in the eczema model and the potential value of TAK-427 as a therapeutic agent for atopic dermatitis. TAK-427 has antihistaminic activity as well as an anti-inflammatory effect, but its inhibitory effect on eczema formation was found to be unrelated to its antihistaminic effect. To elucidate the anti-inflammatory effect of TAK-427, we investigated expression of the mRNA of several proinflammatory cytokines and chemokines in the lesioned skin.

	Materials and Methods

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Animals. Std:Hartley guinea pigs (4-5 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). The animals were housed under controlled temperature (24 ± 1°C) and humidity (55 ± 5%) conditions and given access to food and water ad libitum. The care and use of the animals and the experimental protocol used in this study were approved by the Experimental Animal Care and Use Committee of Takeda Chemical Industries, Ltd. (Osaka, Japan).

Drugs and Materials. Sodium dodecylsulfate and Vaseline (white, high-pure) were purchased from Wako Pure Chemical (Osaka, Japan), and ketotifen fumarate, terfenadine, ovalbumin (grade III; OVA), and dexamethasone were from Sigma-Aldrich (St. Louis, MO). Pontamine sky blue 6B was from Tokyo Kasei Kogyo (Tokyo, Japan). Azelastine hydrochloride was extracted from AZEPTIN tablets (Eisai, Tokyo, Japan). TAK-427 (Fig. 1) and cetirizine were synthesized at Takeda Chemical Industries, Ltd. All drugs were suspended in a 5% gum arabic solution.

Epicutaneous Sensitization and Challenge. Female guinea pigs were anesthetized by intramuscular injection of a combination of ketamine (Sankyo, Tokyo, Japan) and xylazine (Bayer AG, Leverkusen, Germany), and 10 µg of OVA mixed with 0.5 mg of Al(OH)₃ was injected intradermally at four sites on a shaved shoulder. One week later, a lint patch (3 × 4 cm) spread with an emulsion mixture containing 10% OVA, 10% sodium dodecylsulfate, 30% Vaseline, and 50% water was placed on the shaved shoulder and maintained in position for 48 h by wrapping with cloth adhesive tape. Additional sensitization 3 weeks after the OVA injection was performed in essentially the same manner at same site with emulsion consisting of 10% OVA, 5% SDS, 37% Vaseline, and 48% water. One week after the final sensitization, antigen challenge was carried out by bilateral topical application to a shaved flank of a lint patch (1 × 1 cm) spread with Vaseline or a 10% OVA emulsion containing 20% water and 70% Vaseline. The patches were maintained in position for 24 h by wrapping with cloth adhesive tape and then removed. At 48 h after the challenge, the sites were scored in terms of erythema, edema, and scratch formation, as described in Table 1, and the sum of the three scores was used as the dermatitis score. The drugs were administered to the guinea pigs orally by tube gavage in a volume of 0.2 ml/100 g b.wt. twice daily for 3 days beginning on the day before the OVA challenge.

Histopathological Examinations. The OVA-challenged skin of each animal was punched out (15-mm diameter) 48 h after antigen application, and the specimens were fixed in 10% neutral buffered formalin. After embedding in paraffin, 5-µm sections were stained with hematoxylin-eosin and Biebrich scarlet-iron hematoxylin (Luna stain) to detect infiltrated eosinophils. The skin lesions were scored (1 to 4) for crust formation, epidermal vacuolation, and eosinophil infiltration of the epidermis and the crust. Eosinophils that had infiltrated the dermis were counted in 10 to 12 consecutive high-power fields (200×) of each skin section, and the number per millimeter of length of the epidermal layer was calculated.

Passive Cutaneous Anaphylaxis Reaction. To determine the IgE titer, serum from epicutaneously sensitized guinea pigs was diluted with saline and intradermally injected into the shaved backs of male guinea pigs at a volume of 0.05 ml. Seven days later, the animals were intravenously challenged with 1 ml of 2.5% pontamine sky blue dye containing 2.5 mg of OVA in saline and sacrificed by bleeding 30 min after the challenge. The last dilution to give a threshold reaction (5-mm diameter of blue spot) in two of three guinea pigs was recorded as the OVA-specific IgE titer.

Quantitative PCR. At 24 h after the OVA-challenge, the skin of the challenged sites was collected, and total RNA was extracted with an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's protocol. cDNA was synthesized by incubating 4 µg of total RNA with 33 µl of reaction mixture containing 0.2 µg of pd(N)₆ primer (Amersham Biosciences UK, Ltd., Buckinghamshire, UK) at 37°C for 60 min using Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences UK, Ltd.). The numbers of mRNA copies for IL-8, IL-5, eotaxin, TNF-, GM-CSF, IL-1, IFN-, IL-10, IL-13, and G3PDH were determined based on quantitative PCR standard curves plotted by using data obtained with a sequence detector ABI PRISM 7700 application (Applied Biosystems, Foster City, CA). All primers and probes used in this study were designed with ABI PRISM Primer Express 1.0 software (Applied Biosystems) and were synthesized at Amersham Biosciences UK, Ltd. and PerkinElmer Japan (Yokohama, Japan), respectively. The specificity of the PCR primers was tested under normal PCR conditions in a thermal cycler before quantitative PCR. The probes were labeled with a reporter fluorescent dye, 6-carboxyfluorescein, at the 5'-end and a fluorescent dye quencher, tetramethylrhodamine, at the 3'-end. The absolute copy numbers of cytokine and chemokine mRNA in each sample were calculated based on the standard curve for cDNA (from normal PCR products), and the absolute copies of cytokine mRNA were then normalized against those of G3PDH mRNA. The primers used were G3PDH forward primer (F) 5'-CAAGGTCATCCCAGAGCTGAA-3', reverse primer (R) 5'-CCACAACCGACACATTAGGTG-3', probe 5'-AAGCTCACAGGTATGGCCTTCCGTGTAC-3'; IL-8 (F) 5'-GCTGCGATGCCAGTGTATTAAG-3', (R) 5'-GGTCCACTCTCAATCACTTTCAGT-3', probe 5'-CACACCACACCTTTCCACCCCAAATT-3'; IL-5 (F) 5'-AGCTGCACCTTTTGTAGCCA-3', (R) 5'-CAGAGTTCGATGAGTAGAAAGCAGAG-3', probe 5'-TGTCTGTGTCTGTGCCATCCCCAA-3'; Eotaxin (F) 5'-CAGACAGCCATTGTCTTTGAGA-3', (R) 5'-GCATCCTGAACCCACTTCTTCT-3', probe 5'-AAACCTGACAAAATGATATGTGCGGACCCC-3'; TNF- (F) 5'-CTCATCTACTCCCAGGTCCTCTT-3', (R) 5'-TGATGGCAGAGAGAAGGTTGAC-3', probe 5'-TCCTACCTGCTTCTCACCCATACCGTCA-3'; GM-CSF (F) 5'-CAGTCCTGGAAACACGTGGAT-3', (R) 5'-TCATTCATCACAGCAGCCG-3', probe 5'-CATCAATGAAGCCCTGAGCCTCCTGA-3'; IL-1- (F) 5'-ATGATCCGCTCCACGAGAA-3', (R) 5'-GGATTCCTCTGAGTTTTCGTAGG-3', probe 5'-TGTGGATGAGCCAGTGTCTCCGAA-3'; IFN- (F) 5'-CCATCAAGGAACAAATCATTACTAAGTT-3', (R) 5'-TTTGAATCAGGTTTTTGAAAGCC-3', probe 5'-TTCAAAGACAACAGCAGCAACAAGGTGC-3'; IL-10 (F) 5'-CCCACATGCTTCGAGAGC-3', (R) 5'-ATCCTGTGTTTGGAAGAAAGTCTTC-3', probe 5'-CCGAGCTGCCTTTGGCAGGG-3'; and IL-13 (F) 5'-TCCAACTGCAGCGCCC-3', (R) 5'-GGCCTTGTGCTGGCAAAG-3' probe 5'-CCAGAGGACCCAGAAGATACTGAGCGG-3'. Quantitative PCR was performed with reverse transcription products, TaqMan Universal PCR Mater Mix (Applied Biosystems), forward primer (final, 0.3 µM), reverse primer (final, 0.3 µM), probe (final, 0.2 µM), and distilled water in a total volume of 50 µl. PCR was performed at 50°C for 2 min, at 95°C for 10 min and then for 40 cycles at 95°C for 15 s, and at 60°C for 1 min on the ABI PRISM 7700 detection system.

Statistical Analysis. Statistical analysis of the numbers of infiltrating eosinophils and mRNA levels of cytokines in the skin lesion was performed by a Dunnett's test, and dermatitis manifestations were analyzed by the nonparametric Dunnett's test. Values of P < 0.05 were considered statistically significant. All statistical calculations were performed with the SAS statistical package in our laboratory.

	Results

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Characteristics of OVA-Induced Eczema-Like Skin Reactions in Epicutaneously Sensitized Guinea Pigs. The OVA-specific serum IgE levels of the epicutaneously sensitized guinea pigs measured by the passive cutaneous anaphylaxis reaction titers were 307 ± 48. Topical application of OVA to the epicutaneously sensitized guinea pigs produced eczema-like skin reactions characterized by erythema and edema with scratches in half of the challenge sites (Fig. 2). Histopathological examination 48 h after OVA application revealed that the skin at the OVA-challenged sites was characterized by thickening of the epidermis and inflammatory cell infiltration of both dermis and epidermis (Fig. 3).

View larger version (130K): [in this window] [in a new window]   Fig. 2.   Gross appearance of OVA-induced eczema-like dermatitis in epicutaneously sensitized guinea pigs. OVA was patched for 24 h to shaved flanks of epicutaneously sensitized guinea pigs. Eczema-like dermatitis was induced on both flanks and evaluated at 48 h after OVA application. The animals in the above photograph were three representatives in the control group. Inset: magnification of squared area.

View larger version (158K): [in this window] [in a new window]   Fig. 3.   Histopathological features of OVA-induced eczema-like dermatitis in epicutaneously sensitized guinea pigs. OVA was patched for 24 h to shaved flanks of epicutaneously-sensitized guinea pigs. Skin specimens were collected 48 h after antigen challenge, and histological evaluations were performed as described under Materials and Methods. A and C show normal skin preparations. Significant cellular infiltration seen in the upper dermis of OVA-challenged sites (B). Eczema-like lesions, such as hypertrophy, spongiosis, and acanthosis, were seen in the epidermis of the OVA-challenged sites (D). Staining with hematoxylin-eosin. Bar = 100 µm (A and B) and bar = 50 µm (C and D).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The time course of eosinophil infiltration of the dermis of OVA-induced eczema-like skin lesions in epicutaneously sensitized guinea pigs. OVA or Vaseline was patched for 2 to 24 h to shaved flanks of epicutaneously sensitized guinea pigs. Skin specimens were collected at indicated time after patched challenge. Histological evaluations were performed as described under Materials and Methods. The results are expressed as means ± S.E.M. for five animals.

Effect of TAK-427, Dexamethasone, and Antihistamines on OVA-Induced Eczema-Like Skin Reactions in Epicutaneously Sensitized Guinea Pigs. In animals treated with vehicle, according to the criteria described in Table 1, the scores for erythema, edema, and scratches were 2.41 ± 0.15, 1.91 ± 0.06, and 0.41 ± 0.13, respectively, and the dermatitis score was 4.73 ± 0.25. TAK-427 at doses of 0.3, 3, and 30 mg/kg, p.o. reduced the dermatitis score dose dependently, and statistical significance was observed at 3 mg/kg and above (Table 2). In the animals treated with dexamethasone, the edema and scratch formation in the patched-skin improved, but there was little effect on the erythema. The drug decreased the dermatitis scores, and its effect was significant at doses of 3 and 10 mg/kg (Table 2). On the other hand, the antihistamines azelastine, ketotifen, terfenadine, and cetirizine did not significantly reduce the dermatitis scores (Table 4).

Histopathological Examination of OVA-Induced Eczema-Like Skin Lesions in Epicutaneously Sensitized Guinea Pigs Treated with TAK-427, Dexamethasone, and Antihistamines. In control groups treated with vehicle, the number of eosinophils that had infiltrated the dermis was 138.6 ± 18.7 cells/mm. The eosinophil infiltration of the dermis was significantly inhibited by 40, 54, and 56% with TAK-427 at doses of 0.3, 3, and 30 mg/kg, p.o., respectively (Figs. 5, A and B, and 6A). TAK-427 also dose dependently inhibited eosinophil infiltration into the crust and the epidermal layer, and the effects were significant at the 30 mg/kg dose (Table 4). Vacuolation of epidermis tended to suppress in response to TAK-427, although the effect was not statistically significant (Fig. 5, C and D; Table 4).

View larger version (123K): [in this window] [in a new window]   Fig. 5.   Effects of TAK-427 on eosinophil infiltration and dermal injury at the OVA challenged sites in epicutaneously sensitized guinea pigs. OVA was patched to shaved flanks of epicutaneously sensitized guinea pigs for 24 h. Drugs were administered p.o. twice daily for 3 days beginning on the day before antigen challenge. Skin specimens were collected 48 h after patch challenge with OVA, and histological evaluations were performed as described under Materials and Methods. Representative histologic sections stained with Luna (A and B; bar = 50 µm) or hematoxylin-eosin (C and D; bar = 100 µm; insets, bar = 50 µm) are shown. TAK-427 reduced eosinophil infiltration of the dermis (B; 3 mg/kg, p.o.) and tended to suppress vacuolation (D; 30 mg/kg) at the OVA-challenged sites in epicutaneously sensitized guinea pigs.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of TAK-427 and dexamethasone on eosinophil infiltration of the dermis at OVA-challenged sites in epicutaneously sensitized guinea pigs. OVA was patched for 24 h to shaved flanks of epicutaneously sensitized guinea pigs. Drugs were administered p.o. twice daily for 3 days beginning the day before antigen challenge. Skin specimens were collected 48 h after patch-challenge with OVA, and histological evaluations were performed as described under Materials and Methods. Results are expressed as means ± S.E.M. for 10 to 12 animals. *, p < 0.05; **, p < 0.01 versus control (Dunnett's test).

Messenger RNA Expression of Cytokines in the OVA-Patched Skin of Epicutaneously Sensitized Guinea Pigs Treated with TAK-427 and Dexamethasone. To elucidate the mechanism by which TAK-427 inhibits the development of eczema, we investigated the effect of TAK-427 on expression of the mRNA of cytokines such as IL-13, GM-CSF, IL-1, TNF-, IFN-, IL-8, and IL-10 in the skin at OVA-patched sites. Since in the preliminary experiments mRNA expression of these cytokines was found to be increased by OVA challenge and peak at 6 to 24 h after antigen application, the effect of TAK-427 and dexamethasone was evaluated at 24 h after OVA application. All cytokine mRNA expressions were normalized as the number of copies of cytokine cDNA per copies of G3PDH cDNA (× 10⁵). Expression of IL-13, GM-CSF, IL-1, TNF-, IFN-, IL-8, and IL-10 mRNA in OVA-patched sites increased by 2.5-, 2.7-, 3.1-, 5.1-, 2.5-, 18-, and 10-fold, respectively, compared with the expression in the nonchallenged sites. TAK-427 completely suppressed the mRNA expression of IL-13 and significantly inhibited the mRNA expressions of GM-CSF, IL-1, and IL-8 by 72, 70, and 63%, respectively. TAK-427 clearly inhibited the mRNA expression of IFN- and TNF-, although the differences were not statistically significant, but it did not inhibit IL-10 mRNA expression (Fig. 7). Treatment with dexamethasone significantly inhibited mRNA expression of IL-13, GM-CSF, IL-1, TNF-, IFN-, and IL-8 in the OVA-challenged skin site but not that of IL-10 (Fig. 7). These results indicated that both TAK-427 and dexamethasone inhibited the expression of proinflammatory cytokines after antigen challenge but did not affect the anti-inflammatory cytokine IL-10.

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Effects of TAK-427 and dexamethasone on cytokine mRNA expression at the OVA-challenged sites in epicutaneously sensitized guinea pigs. OVA were patched for 24 h to shaved flanks of epicutaneously sensitized guinea pigs. TAK-427 (30 mg/kg) and dexamethasone (10 mg/kg) were administered p.o. twice daily for 2 days beginning the day before antigen challenge. Skin specimens were collected 24 h after patched challenge with OVA, and cytokine mRNA expression was evaluated as described under Materials and Methods. Results are expressed as relative quantity of mRNA based on G3PDH (× 105) expression, and the data are means ± S.E.M. for 10 animals. Dex: dexamethasone. , p < 0.05; , p < 0.01 versus no patched control (Student's t test); *, p < 0.05; **, p < 0.01 versus control (Dunnett's test).

	Discussion

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The results of this study showed that topical antigen challenge by patch induced eczema-like lesions at the challenged sites in epicutaneously sensitized guinea pigs and that TAK-427 mitigated the development of eczema and suppressed expression of proinflammatory cytokine/chemokine mRNA in this experimental model. Histopathologically, the antigen-challenged sites showed erythema with papules, epidermal hypertrophy, vacuolation of epidermal cells, and cellular infiltration. These results indicated that the eczema model in the guinea pig shares several features with the skin lesions in the acute phase of atopic dermatitis, suggesting that epicutaneous sensitization and challenge play an important role in the development of eczema at the local sites. This notion is supported by two recent studies in which application of the antigen (OVA) to mouse skin resulted in the induction of antigen-specific IgE antibodies and an eczema-like skin response (Wang et al., 1996; Spergel et al., 1998, 1999).

In addition, the eczema model in the guinea pig showed similarities to atopic dermatitis pharmacologically because dexamethasone was effective in suppressing dermal inflammation and eczema formation, but none of the four antihistamines tested (azelastine, ketotifen, terfenadine, and cetirizine) were effective. The highest dosage levels of antihistamines used in this study were about 10 times the ID₅₀ values for their antihistaminic effects, and since they were ineffective against the dermal lesions, histamine does not seem to play an important role in terms of the development of eczema in this model. These observations suggest that the guinea pig model may be a useful tool for evaluating the potency of drugs for the treatment of atopic dermatitis.

TAK-427, a novel imidazopyridazine derivative, was discovered as a result of testing a number of compounds for anti-inflammatory activity in the guinea pig model of eczema and its antihistaminic activity in vivo and in vitro, and it is currently under development as a new therapeutic agent for atopic dermatitis. In this eczema model, TAK-427 significantly reduced the manifestations of dermatitis and eosinophil infiltration of the dermis and epidermis at the OVA-patched sites. Although TAK-427 displays antihistaminic activity (ID₅₀, approximately 1 mg/kg), its inhibitory effect on the development of eczema is not attributable to antihistaminic effect because none of the antihistamines exerted an inhibitory effect in this experimental model.

Evidence of eosinophil infiltration and presence of eosinophil-derived major basic protein in the dermal layer have been well documented in atopic dermatitis, although the actual role of eosinophils in the pathogenesis of atopic dermatitis is poorly understood. Eosinophil major basic protein and eosinophil cationic protein are known to have a cytotoxic effect (Rothenberg, 1998), and a recent study indicated that the absence of eosinophils in OVA-sensitized skin sites of IL-5-deficient mice was associated with a lack of increase in the thickness of the epidermis and dermis, a common feature of the skin lesions in atopic dermatitis. These results suggest that eosinophils play an important role in the cutaneous hypertrophy in atopic dermatitis (Spergel et al., 1999), and inhibition of eosinophil infiltration may improve dermal symptoms in atopic dermatitis. The inhibitory effect of TAK-427 and dexamethasone on eosinophil infiltration may be related to their amelioration of the manifestations of dermatitis in the guinea pig model.

The effect of cetirizine on eosinophil infiltration has been a matter of controversy. Some groups have reported finding that cetirizine is effective in inhibiting eosinophil infiltration/migration into human skin in allergic conditions (Fadel et al., 1987; Charlesworth et al., 1989b), whereas other groups have reported that late-phase reactions, including eosinophil infiltration, are unaffected by cetirizine (Varney et al., 1992; Atkins et al., 1997; Zweiman et al., 1997). In our eczema model, cetirizine aggravated the eosinophil infiltration at OVA-patched skin sites rather than suppressing it. The reason for the difference in the findings in these studies is unclear.

The skin at the site of the lesions in atopic dermatitis hyperexpressed several proinflammatory cytokines and chemokines, including IL-4, IL-5, IFN-, IL-13, GM-CSF, TNF-, IL-1, IL-8, and eotaxin, and the anti-inflammatory cytokine IL-10 (Van Joost et al., 1992; Hamid et al., 1994; Ohmen et al., 1995; Pastore et al., 1997; Van der Ploeg et al., 1997; Yawalkar et al., 1999). IFN- has been reported to be important for the development of skin hypertrophy in a murine model of dermatitis (Carroll et al., 1997; Spergel et al., 1999). GM-CSF, IL-13, TNF-, and IL-8 works as a potent stimulant of the recruitment of inflammatory cells (Nakajima et al., 1994; Erger and Casale, 1995; Pastore et al., 1997; Ying et al., 1997). The present study has demonstrated that the OVA-patched skin sites also hyperexpressed IFN-, IL-13, GM-CSF, TNF-, IL-1, IL-8, and IL-10. We think that the cytokines act together to produce the dermal inflammation and eczema-like lesions in the guinea pig model. It is well known that eotaxin and IL-5 are one of the most related cytokine/chemokine to eosinophil infiltration, but their mRNA expression did not increase at the sites of skin lesions in this guinea pig eczema model (data not shown), suggesting that local expression of eotaxin and IL-5 dose not play a predominant role in the eosinophil infiltration in this model.

In this study, TAK-427 was found to significantly suppress expression of mRNA of IL-8, GM-CSF, IL-13, and IL-1 and tends to suppress expression of the mRNA of IFN- (p = 0.0657) and TNF- (p = 0.0639). Dexamethasone also inhibited mRNA expression of these proinflammatory cytokines. Neither TAK-427 nor dexamethasone, however, suppressed mRNA expression of the anti-inflammatory cytokine IL-10. Similar results were observed in atopy patch test lesions treated with topical glucocorticoids (Langeveld-Wildschut et al., 2000). Since we have observed that TAK-427 has no effect on T or B cell proliferation in vitro in mice, it is not a general immunosuppressant (unpublished data). The inhibition of mRNA expression of proinflammatory cytokines may have contributed to the anti-inflammatory effects of TAK-427 in this experimental model. Although the precise mechanism remains to be elucidated, since TAK-427 has the inhibitory effect of TNF- release from the mast cell by IgE-dependent mechanism (in preparation), this mechanism may have contributed to the inhibition of mRNA expression of proinflammatory cytokines. As T cells are one of the most important source of cytokines, the effect of TAK-427 on T cells migration into the challenged sites are now investigated.

In summary, the eczema-like skin lesions in this experimental model exhibit several features similar to those of the acute phase of atopic dermatitis in terms of manifestations of dermatitis, dermal inflammation, and epidermal injury, suggesting that this guinea pig model may be a useful tool for evaluating the potency of drugs for the treatment for atopic dermatitis. TAK-427 suppressed the allergic dermal inflammation that leads to eczema formation by inhibiting the expression of proinflammatory cytokines at the lesion sites.